Novel mixture and compounds from mycelia of antrodia camphorata and use thereof

ABSTRACT

The present invention relates to a compound derived from mycelium of  Antrodia camphorata . The present invention also relates to the composition or mycelium comprising the compounds of the invention. The composition of the invention decreases systolic blood pressure and increases high density lipoprotein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continue-in-part Application of U.S. patentapplication Ser. No. 12/840,655, filed Jul. 21, 2010, which is aContinue-in-part Application of U.S. patent application Ser. No.12/078,985, filed Apr. 9, 2008, which is a Divisional Application ofU.S. application Ser. No. 11/312,480, filed Dec. 21, 2005, which iscurrently pending, the entire contents of which are incorporated hereinby reference, which is a Divisional Application of U.S. application Ser.No. 10/793,820, filed Mar. 8, 2004, now U.S. Pat. No. 7,109,232, issuedSep. 19, 2006, the entire contents of which are incorporated herein byreference.

Although incorporated by reference in its entirety, no arguments ordisclaimers made in the parent application apply to thisContinuation-in-part application. Any disclaimer that may have occurredduring the prosecution of the above-referenced application(s) is herebyexpressly rescinded. Consequently, the Patent Office is asked to reviewthe new set of claims in view of the entire prior art of record and anysearch that the Office deems appropriate.

FIELD OF THE INVENTION

The present invention relates to novel mixture and compounds frommycelium of Antrodia camphorata and the use thereof. The presentinvention relates to the composition or mycelium comprising thecompounds of the invention.

BACKGROUND OF THE INVENTION

The fruiting body of Antrodia camphorata (Polyporaceae, Aphyllophorales)is well known in Taiwan as a traditional Chinese medicine. It grows onlyon the inner heartwood wall of the endemic evergreen Cinnamomunkanehirai (Hay) (Lauraceae) in Taiwan. It is rare and has not beencultivated. The fruiting bodies have been used for treating of food anddrug intoxication, diarrhea, abdominal pain, hypertension, itchy skin,and liver cancer. Very few biological activity studies have beenreported hitherto.

Antrodia camphorata also known as “niu-chang-chih” or “niu-chang-ku” inTaiwan, was recently reported as a new fungus species characterized bythe cylindrical shape of its basidiospores appearing in fruiting bodies,weakly amyloid skeletal hyphae, bitter taste and light cinnamonresupinate to pileate basidiocarps, as well as chlamydospores andanthroconidia in pure culture. The growth of this new fungus species isextremely slow and restricted to an endemic tree species, Cinnamomumkanehirai Hay (Lauraceae), as the only host. The detailedcharacterization and taxonomic position of Antrodia camphorata weredescribed in Wu, S.-H., et al., Antrodia cinnamomea (“niu-chang-chih”),New combination of a medicinal fungus in Taiwan, Bot. Bull. Acad. Sin.38: 273-275 (1997).

In Taiwanese folk medicine, the fruiting bodies of Antrodia camphorataare believed to have certain medical effects. According to thetraditional way, the fruiting bodies are ground into dry powder orstewed with other herbal drugs for oral uptake to treat conditionscaused by poisoning, diarrhea, abdominal pain, hypertension, skin itchesand liver cancer. However, little pharmacological or clinical study inthese aspects has appeared in literature to date. Because of thestringent host specificity and rarity in nature, as well as the failureof artificial cultivation, “niu-chang-chih” is very expensive in Taiwan.In recent years, the fruiting bodies of this fungus with high qualityhave been sold at an extremely high price of around U.S. $ 15,000 perkg.

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to provide novelmixture from mycelium of Antrodia camphorata.

Another object of the present invention is to provide novel compoundsfrom mycelium of Antrodia camphorata.

Further object of the present invention is to provide novel compositioncomprising the compounds of the invention.

Further object of the present invention is to provide novel mycelium ofAntrodia camphorata comprising the compounds of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows HMBC correlations of compound 2.

FIG. 2 shows the compounds of the invention.

FIG. 3 shows NOE (nuclear Overhauser effect) correlations of compounds 4and 5 of the invention.

FIGS. 4A-D shows test results of compound 3 of the invention.

FIGS. 5A-C shows test results of ACM (Antrodia camphorata myceliapowder) H₂O Extract.

FIGS. 6A-F shows test results of ACM EtOH (ethyl alcohol) Extract.

FIGS. 7A-E shows test results of compound 1 of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound having the formula

wherein R₁ is C₁₋₁₀ alkyloxy, C₂₋₁₀ alkenyloxy, or C₂₋₁₀ alkynyloxy; andR₂ is H, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl or C₂₋₁₀ alkynyl, provided that ifR₁ is C₁₋₁₀ alkyloxy, R₂ is not H, and if R₁ is C₁ alkyloxy, R₂ is notC₁ alkyl.

In the compound of the invention, the preferred R₁ is C₂₋₆ alkenyloxy,or C₂₋₆ alkynyloxy; the more preferred R₁ is C₂₋₆ alkenyloxy substitutedwith C₁₋₆ alkyl and the most preferred R₁ is butenyloxy substituted withmethyl. In the compound of the invention, the preferred R₂ is C₁₋₆alkyl, the most preferred R₂ is isobutyl.

Accordingly, the preferred compound of the invention is

-   3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-dione,-   3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-2,5-dione,-   3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-1-ol-2,5-dione,-   3R*,4S*-1-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolidine-2,5-dione,    or-   3R*,4R*-1-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolidine-2,5-dione.

The further preferred compound of the invention is

-   3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-2,5-dione    or-   3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-1-ol-2,5-dione.

The further preferred compound of the invention is

-   3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-1-ol-2,5-dione.

The present invention also provides a mixture from mycelium of Antrodiacamphorata, which comprises the compound of the invention. The mixtureof the invention is prepared from water or organic solvent extract ofmycelium of Antrodia camphorata. The organic solvent includes but is notlimited to alcohol (such as CH₃OH, C₂H₅OH, C₃H₇OH), ester (such asacetyl acetate), alkane (such as hexane) and halogenated alkane (such asCH₃Cl, C₂H₂Cl₂). The preferred organic solvent is ethanol or alcoholicsolvent without causing any side effect of human. The mixture of theinvention can decrease systolic blood pressure or increase high densitylipoprotein. In addition, the same mixture has central cholinergicagonism, hepatoprotection, anti-inflammation or anti-tumor activity.Especially, the mixture of the invention can inhibit tumor from thecells or tissues selected from the group consisting of liver, intestine,bone, blood, lymph and breast. The subject accepting the mixture of theinvention includes but is not limited to human, mammal, mouse, rat,horse, pig, chicken, duck, dog and cat.

The present invention also provides a composition, which comprises thecompound of the invention. The composition of the invention can decreasesystolic blood pressure or increase high density lipoprotein. Inaddition, the composition of the invention has central cholinergicagonism, hepatoprotection, anti-inflammation or anti-tumor activity.Especially, the composition of the invention can inhibit tumor from thecells or tissues selected from the group consisting of liver, intestine,bone, blood, lymph and breast. The subject accepting the composition ofthe invention includes but is not limited to human, mammal, mouse, rat,horse, pig, chicken, duck, dog and cat.

The present invention also provides novel mycelium of Antrodiacomprising the compounds of the invention. The preferred mycelium has atleast 1% of the weight of raw mycelium being the total weight of thecompounds 1-5 of the invention. The most preferred mycelium has at least3% of the weight of raw mycelium being the total weight of the compounds1-5 of the invention. The mycelium of Antrodia camphorata is previouslyprepared according to submerged liquid fermentation such as T. L. M.Stamford et al., Food Science “Protein enrichment of cashew wastes foranimal feeds” from http://www.unu.edu/unupress/food/8F101e/8F101E0b.htm.

Example

The examples below are non-limiting and are merely representative ofvarious aspects and features of the present invention.

General Experimental Procedures

Melting points were measured on a Yanagimoto micro hot-stage meltingpoint apparatus and uncorrected. Optical rotations were measured with aJasco DIP-360 automatic polarimeter. UV spectra were measured with aShimadzu UV-2200 recording spectrophotometer. IR spectra were measuredwith a Jasco FT/IR-230 infrared spectrometer. ¹H- and ¹³C-NMR spectrumwere measured with a Varian Unity Plus 500 spectrometer. EIMS andHR-EIMS were measured with a Jeol JMS-AX 505 HAD mass spectrometer at anionization voltage of 70 eV. Column chromatography was carried out onsilica gel BW-820 MH (normal phase) and Chromatorex-ODS DM1020T(reversed phase) (Fuji Silysia).

Extraction and Isolation

Antrodia camphorata mycelia powder (ACM) (60 g), from Simpson BiotechCo. Ltd., Taiwan, October 2001, were three times extracted with CHCl₃for 3 h under reflux. The CHCl₃ extract (5.3 g) was chromatographed onsilica gel eluted with n-hexane-acetone (19:1-14:6), and CHCl₃-MeOH(1:1) to give nine fractions (Fr. 1-9). Fraction 2 was chromatographedon silica gel to give compound 1 (8.7 mg). Fraction 4 waschromatographed on normal and reversed phase silica gel to give compound2 (13.6 mg). Fraction 5 was chromatographed on silica gel eluted withn-hexane-acetone (8:2) to give ergosterol peroxide (35.8 mg). Fraction 6gave compound 3 (14.6 mg) by combination of normal and reversed phasesilica gel column chromatography. Fraction 7 yielded a mixture ofcompounds 4 and 5 (4:1) by column chromatography. The mixture ofcompounds 4 and 5 were subsequently separated by preparative HPLC[column: Tosoh TSK-gel ODS-80™ (21.5.times.300 mm), mobile phase: CH₃OH—H₂O containing 0.1% TFA (70:30)].

3-Isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-dione (compound1): yellow oil; UV (MeOH) λ_(max) (log ε) 227 (4.1), 258 (3.9), 275(3.8), 355 (3.4) nm; IR (CHCl₃) ν_(max) 1763 cm⁻¹; ¹H-NMR TABLE 1;¹³C-NMR TABLE 2; EIMS m/z 314 [M]⁺(100), 246 (100), 131 (100); HR-EIMSm/z 314.1523 (Calcd for C₁₉H₂₂O₄, 314.1518).

3-Isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrole-2,5-dione (2):yellow needles (n-hexane-AcOEt); mp 110-111° C.; UV (MeOH) λ_(max) (logε) 230 (4.3), 272 (3.5), 355 (3.7) nm; IR (CHCl₃) ν_(max) 1724 cm⁻¹;¹H-NMR TABLE 1; ¹³C-NMR TABLE 2; EIMS m/z 313 [M]⁺(8), 245 (100), 203(77), 131 (28); HR-EIMS m/z 313.1681 (Calcd for C₁₉H₂₃NO₃, 313.1678).

X-ray Crystallography of Compound 2:

Yellow needles were obtained by crystallization from n-hexane-AcOEt andselected for data collection. Crystal data: C₁₉H₂₃ NO₃; M_(r)=313.40;dimensions 0.15×0.02×0.02 mm; triclinic, space group P1 (#2), a=6.3505(5) Å, b=12.205 (1) Å, c=12.560 (2) Å, α=64.623(7)°, β=75.358(4)°,γ=84.681(5)°, V=850.9(2) Å³, Z=2, D_(calc)=1.223 g/cm³, μ(MoKα)=0.82cm⁻¹, F₀₀₀=336.00. Measurement was made on a Rigaku RAXIS-RAPID ImagingPlate diffractometer with graphite monochromated Mo—Kα (λ=0.71069 A)radiation at 93 K. Of the 8950 reflections which were collected, 4745were unique (R_(int)=0.108); equivalent reflections were merged. Thecrystal structure was solved by direct methods (SHELXS86) and refined byfull-matrix least-squares. The non-hydrogen atoms were refinedanisotropically. Hydrogen atoms were included but not refined. The finalindices were R=0.074, R_(w)=0.099, with GOF (Guest ObserverFacility)=1.06. The maximum and minimum peaks on the final differenceFourier map corresponded to 0.83 and -0.89 e⁻/Å³, respectively.

3-Isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-1-ol-2,5-dione(Compound 3): yellow oil; UV (MeOH) λ_(max) (log ε): 232.5 (4.3), 296(3.7), 374 (3.7) nm; IR (CHCl₃) ν_(max) 1717 cm⁻¹; ¹H-NMR TABLE 1;¹³C-NMR TABLE 2; EIMS m/z 329 [M]⁺(12), 261 (100), 131 (50); HR-EIMSm/z: 329.1637 (Calcd for C₁₉H₂₃NO₄, 329.1627).

3R*,4S*-1-Hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolidine-2,5-dione(4): colorless oil; [α]_(D) ²³+2.5° (c 0.2, MeOH); UV (MeOH) λ_(max)(log ε): 225 (4.3), 275 (3.3), 283 (3.2) nm; IR (CHCl₃) ν_(max) 1715cm⁻¹; ¹H-NMR TABLE 1; ¹³C-NMR TABLE 2; EIMS m/z 331 [M]⁺(2), 263 (67),207 (66), 191 (30), 179 (40), 133 (64), 69 (100); HR-EIMS m/z 331.1747(Calcd for C₁₉H₂₅NO₄, 331.1783).

3R*,4R*-1-Hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolidine-2,5-dione(5): colorless oil; [α]_(D) ²³+3.0° (c 0.2, MeOH); UV (MeOH) λ_(max)(log ε): 227 (4.3), 275 (3.4), 283 (3.3) nm; IR(CHCl₃) ν_(max) 1715cm⁻¹; ¹H-NMR TABLE 1; ¹³C-NMR TABLE 2; EIMS m/z 331 [M]⁺ (1), 263 (45),207 (50), 191 (75), 179 (30), 133 (100), 69 (92); HR-EIMS m/z 331.1766(Calcd for C₁₉H₂₅NO₄, 331.1783).

Ergosterol peroxide: colorless needles (n-hexane-acetone); mp 165-169°C. (lit² mp 171-174° C.).

Cytotoxic Assays. The in vitro LLC tumor cell assay was carried out bysulforhodamin B (SRB) method. The 50% growth inhibition (ED₅₀) wascalculated by Probit method.

Results and Discussion

The CHCl₃ extract of the mycelium of Antrodia camphorata was repeatedlychromatographed on normal and reversed phase silica gel to afford fivenew maleic and succinic acid derivatives (compounds 1-5) together withergosterol peroxide.

TABLE 1 ¹H-NMR Spectral Data of Compounds 1-5 (δ ppm, J = Hz) (500 MHz,CDCl₃) H 1 2 3 4 5 3 — — — 2.87 (1H, m) 3.08 (1H, m) 4 — — — 3.52 (1H,d, 4.07 (1H, d, J = 4.0) J = 8.0) 1′ 2.59 (2H, d, 2.51 (2H, d, 2.50 (2H,d, 1.51 (1H, m) 1.02 (1H, m) J = 7.0) J = 7.0) J = 7.0) 1.72~1.84 (1H)1.42~1.48 (1H) 2′ 2.12 (1H, 2.06 (1H, 2.05 (1H, 1.72~1.84 (1H) 1.42~1.48(1H) sep, J = 7.0) sep, J = 7.0) sep, J = 7.0) 3′ 0.94 (6H, d, 0.90 (6H,d, 0.88 (6H, d, 0.70 (3H, d, 0.66 (3H, d, J = 7.0) J = 7.0) J = 7.0) J =6.5) J = 6.5) 4′ 0.89 (3H, d, 0.80 (3H, d, J = 6.5) J = 6.5) 2″, 6″ 7.50(2H, d, 7.50 (2H, d, 7.50 (2H, d, 7.07 (2H, d, 6.96 (2H, d, J = 9.0) J =9.0) J = 9.0) J = 8.5) J = 9.0) 3″, 5″ 7.02 (2H, d, 6.95 (2H, d, 6.98(2H, d, 6.87 (2H, d, 6.84 (2H, d, J = 9.0) J = 9.0) J = 9.0) J = 8.5) J= 9.0) 1′″ 4.57 (2H, d, 4.56 (2H, d, 4.55 (2H, d, 4.47 (2H, d, 4.47 (2H,d, J = 6.6) J = 6.5) J = 6.9) J = 6.5) J = 6.5) 2′″ 5.50 (1H, 5.50 (1H,5.49 (1H, 5.47 (1H, brt, 5.47 (1H, brt, brt, J = 6.6) brt, J = 6.5) brt,J = 6.9) J = 6.5) J = 6.5) 4′″ 1.81 (3H, s) 1.81 (3H, s) 1.81 (3H, s)1.79 (3H, s) 1.79 (3H, s) 5′″ 1.76 (3H, s) 1.76 (3H, s) 1.76 (3H, s)1.73 (3H, s) 1.73 (3H, s)

TABLE 2 ¹³C-NMR Spectral Data for Compound 1-5 (δ ppm) (125 MHz, CDCl₃)C 1 2 3 4 5 2 166.4 (s) 171.7 (s) 168.8 (s) 174.8 (s) 175.1 (s) 3 139.8(s) 138.8 (s)^(a)) 135.9 (s)^(a))  44.6 (d)  40.3 (d) 4 140.2 (s) 139.2(s)^(a)) 136.0 (s)^(a))  49.8 (d)  47.5 (d) 5 165.4 (s) 171.1 (s) 168.1(s) 173.2 (s) 173.6 (s) 1′  33.6 (t)  32.8 (t)  33.2 (t)  40.4 (t)  35.3(t) 2′  27.9 (d)  28.1 (d)  28.4 (d)  25.3 (d)  25.2 (d) 3′  22.7 (q) 22.7 (q)  23.0 (q)  21.3 (q)  21.8 (q) 4′  23.0 (q)  22.4 (q) 1″ 119.9(s) 121.2 (s) 120.8 (s) 127.9 (s) 125.1 (s) 2″, 6″ 131.1 (d) 130.9 (d)131.0 (d) 128.8 (d) 130.2 (d) 3″, 5″ 115.1 (d) 114.9 (d) 115.0 (d) 115.4(d) 115.0 (d) 4″ 160.9 (s) 160.1 (s) 160.2 (s) 158.7 (s) 158.7 (s) 1′″ 65.0 (t)  64.9 (t)  65.1 (t)  64.1 (t)  64.8 (t) 2′″ 118.7 (d) 119.3(d) 119.2 (d) 119.4 (d) 119.3 (d) 3′″ 139.1 (s) 138.6 (s)^(a)) 138.9 (s)138.3 (s) 138.4 (s) 4′″  25.2 (q)  25.8 (q)  26.1 (q)  25.8 (q)  25.8(q) 5′″  18.2 (q)  18.2 (q)  18.5 (q)  18.1 (q)  18.2 (q)^(a))Assignments may be interchangeable.

The structures of the new compounds were determined as follows: Compound2 gave yellow needles, mp 110-111° C., and the molecular formulaC₁₉H₂₃NO₃ was assigned by HR-EIMS. The IR spectrum showed an imidecarbonyl absorption at 1724 cm⁻¹. The ¹³C-NMR spectrum showed signals offour methyl carbons, two methylene carbons, and one methine carbon inthe aliphatic region, as well as one benzene ring, one olefinic groupand two carbonyl carbons. The ¹H-NMR spectrum showed the presence of anisobutyl moiety at δ 0.90, 2.06, and 2.51, a 3-methyl-2-butenyloxymoiety at δ 1.76, 1.81, 4.56, and 5.50, and a para-substituted benzenemoiety at δ 6.95 and 7.50, which was further supported by ¹H-¹H COSY(cooler synchrotron) and HMQC (heteronuclear multiple quantum coherence)experiments. Long range correlations were observed by HMBC as shown inFIG. 1. On the basis of the molecular formula and the ¹³C-NMR spectrum,this compound was deduced to contain further CHNO atoms, including onemore carbonyl carbon. Thus, this ambiguous part was speculated to be amaleimide group. This structure was then established to be3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrole-2,5-dione byX-ray analysis.

The molecular formula of compound 1 was assigned as C₁₉H₂₂O₄ by HR-EIMS.The IR spectrum revealed a carbonyl absorption of acid anhydride at 1763cm⁻¹. The ¹H-NMR spectrum of compound 1 was similar to that of compound2, and showed the presence of an isobutyl moiety, a3-methyl-2-butenyloxy moiety, and a para-substituted benzene ring. Fromthe HMBC spectrum, compound 1 was demonstrated to have the same partialstructure to compound 2 (FIG. 1), in which the presence of a maleicanhydride group was deduced on the basis of the molecular formulacompound 1 was consequently determined as3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-dione.

The molecular formula of compound 3 was assigned as C₁₉H₂₃NO₄ byHR-EIMS. The IR spectrum showed a carbonyl absorption at 1717 cm⁻¹,assignable to a hydroxy imide. The ¹H- and ¹³C-NMR spectra were alsosimilar to those of compounds 1 and 2, and showed the presence of anisobutyl moiety, a 3-methyl-2-butenyloxy moiety, and a para-substitutedbenzene ring. In the HMBC experiment, compound 3 was shown to have thesame partial structure as compound 2 (FIG. 1). Compound 3 contains onemore oxygen atom than compound 2, therefore, this compound wasdetermined to be(3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl-1H-pyrrol-1-ol-2,5-dione.

Compounds 4 and 5 had the same R_(f) values and the same molecularformula by HR-EIMS (C₁₉H₂₅NO₄, found 331.1747 and 331.1766,respectively), however, they could be separated by preparative HPLC. TheIR spectrum of both compounds showed a hydroxy imide carbonyl absorptionat 1715 cm⁻¹. In the ¹H- and ¹³C-NMR spectra, both compounds showed thepresence of an isobutyl moiety, a 3-methyl-2-butenyloxy moiety, and apara-substituted benzene ring, but the isobutyl methylene protonsdisplayed a multiplet and not a doublet as for compounds 1-3. The ¹H-¹HCOSY spectrum indicated that this methylene group is attached to a—CH—CH— unit. The ¹³C-NMR spectra of compounds 4 and 5 exhibited twoadditional sp³ carbon signals, replacing two sp² carbon signals observedfor compounds 1-3. Therefore, compounds 4 and 5 were not N-hydroxymaleimides, but rather N-hydroxy succinimides, with stereocenters atpositions C-3 and C-4 in the succinimide ring.

Compounds 4 and 5 were determined to be trans and cis isomers,respectively, from the coupling constant between H-3 and H-4 (4.0 and8.0 Hz for compounds 4 and 5, respectively). No NOE was observed betweenH-3 and H-4 in the NOESY (Nuclear Overhauser Effect Spectroscopy)spectrum of compound 4, while appreciable NOE was observed in that ofcompound 5. The optical rotations of compounds 4 and 5 showed +2.5° and+3.0°, respectively, while their CD spectra showed no Cotton effects atany wave length, suggesting that both compounds 4 and 5 are racemicmixtures. Resolution of these racemic mixtures by HPLC using a chiralcolumn with several solvent systems was unsuccessful. At present, wecannot definitely conclude whether these compounds are optically activecompounds or racemic mixtures. Thus, their relative structures weredetermined as 3R*,4S*- and3R,4R*-1-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolidine-2,5-dione,respectively.

Isolation of these type of maleic and succinic acid derivatives fromnature were second time followed by the report of Aquveque et al.

The cytotoxic activity of the chloroform extract and isolated compoundswere investigated using LLC (Lewis lung carcinoma) cell line (TABLE 3).The chloroform extract showed moderate cytotoxic effects with an ED₅₀value of 26.7 μg/ml. Maleic compounds 1 and 4 had no cytotoxic activity,whereas compounds 2 and 3 were found to be cytotoxic to the LLC cellline with ED₅₀ values lower than that of the chloroform extract.

TABLE 3 50% Growth Inhibition (ED₅₀) Values of the CHCl₃ Extract andCompounds 1-4 from the mycelia of Antrodia camphorata against LLC CellLine ED₅₀ (μg/ml) CHCl₃ extract 26.7 1 >20 2 3.6 3 7.5 4 >10Adriamycin^(a)) 0.14 ^(a))Positive control.Tumor Assay of ACM (Antrodia camphorata Mycelia Powder)

A. Cell Line

-   -   Adherent Cell        -   MCF-7: human breast carcinoma        -   HT-29: human colon adenocarcinoma        -   KATO III: human stomach carcinoma        -   SW480: human colon adenocarcinoma        -   SW620: human colon adenocarcinoma        -   HepG2: human liver carcinoma    -   Suspension Cell        -   EL4: mice lymphoma

B. Samples

-   -   Compound 1, Compound 3, ACM EtOH Extract, ACM H₂O Extract

C. Assay Method

-   Calculate ED₅₀ (50% inhibition of effective dose)-   Adherent cell: MTT (methyl thiazolyl tetrazolium) method for MCF-7,    HT-29, KATO III, SW480, HepG2, cells are determined at 3 days. SW620    at 4 days-   Suspension cell: Cell count method; EL4 cells count at 5 days

D. Result

Calculation: y=m Ln(x)+b

Example

X Y 0 0.97 10 ppm 0.941 30 ppm 0.6 100 ppm  0.331

Use value of X (10, 30, 50 ppm) and Y to get correlation curve

y=−0.2643 Ln(x)+1.5321

ED₅₀=exp[0.97/2−1.5321/(−0.2643)]

Sample Preparation and Sample Description A. ACM (Antrodia CamphorataMycelia Powder) H₂O Extract

1. Add 1 g of ACM into 40 ml of RO H₂O in a 250 ml beaker, put thebeaker in ultrasonic water bath for 20 min at room temperature2. Stir at 45° C. water bath for 45 min3. Place the beaker in ultrasonic water bath for another 20 min4. Centrifuge the sample at 3000 rpm for 15 min5. Collect supernatant and perform serial dilutions with media

B. Determination of Sample Concentration

1. Weigh an evaporating dish (W1)2. Add 10 ml of H₂O extract sample in the evaporating dish3. Place the evaporating dish in the oven to remove water (W2)

Sample weight/ml=(W2−W1)/10

C. ACM (Antrodia Camphorata Mycelia Powder) EtOH Extract

1. Add 100 ml of 95% alcohol to 20 g ACM in a 500 ml beaker and stir for10 min at room temperature2. Filter the suspension through Advantec Number 1 filter paper, andcollect the filtrate3. Concentrate the filtrate by rotary vacuum evaporator to removealcohol.D. Compound 1: Pure compound from ACME. Compound 3: Pure compound from ACM

MTT Assay Method

1. Discard old media after cell proliferation, then wash cells once withphosphate-buffered saline (PBS) once2. Wash down the cells with trypsin-EDTA3. Centrifuge at 1200 rpm for 5 min, then discard supernatant4. Suspend the pellet with 10 ml medium5. Mix 100 μl cell suspension with 100 μl trypan blue to calculateviable cells6. Add 1×10⁴ cells/100 μl medium in each well of the 96 well plate,incubate the plate in CO₂ incubator at 37° C. for 24 hrs7. Discard old medium, wash cells once with PBS8. Add 100 μl sample in each well, incubate the plate in CO₂ incubatorat 37° C.9. Wash cells once with PBS at 3rd, 4th and 5th days,10. Add 57 μl MTT (0.88 mg/ml) in each well11. After 4 hrs discard MTT and wash cells with PBS once

12. Add 50 μl DMSO/Well

13. Read at OD545 on Elisa reader

Cell Count Method (EL4 Cell Line)

1. Discard old media after cell proliferation by centrifugation2. Resuspend the pellet with fresh medium3. Mix 100 μl cell suspension with 100 μl trypan blue to calculateviable cells4. Prepare different concentration of samples that contain 1×10⁵ cell/mlsample5. Load 100 μl sample in each well of the 96 well plate, incubate theplate at 37° C. CO₂ incubator6. Calculate viable cells at 3rd, 4th and 5th days

PBS

NaCl   8 g KCl 0.2 g Na₂HPO₄ 1.4 g KH₂PO₄ 0.2 g Make volume to 1 L PH7.4

Result and Discussion

ED₅₀ of ACM on Cell Lines

KATO Cell line HepG2 HT-29 III EL4 SW480 SW620 MCF-7 Compound 1 21 ppm52 ppm 38 ppm 3.5 ppm 15 ppm 6 ppm Compound 3 35 ppm 42 ppm 69 ppm 2.6ppm 20 ppm 27 ppm 0.02 ppm ACM EtOH 32 ppm 52 ppm 156 ppm 2.6 ppm 71 ppm4 ppm Extract ACM H₂O 295 ppm 707 ppm 20 ppm 207 ppm 132 ppm 318 ppmExtract

Detailed Test Results as Follows:

Compound 3 of the invention: HepG2 (FIG. 4A), EL4 (FIG. 4B), HT-29 (FIG.4C) and Kato III (FIG. 4D).

ACM H₂O Extract: HepG2 (FIG. 5A), SW620 (FIG. 5B) and EL4 (FIG. 5C). ACMEtOH Extract: HT-29 (FIG. 6A), SW480 (FIG. 6B), SW620 (FIG. 6C), EL4(FIG. 6D), HepG2 (FIG. 6E) and Kato III (FIG. 6F).

Compound 1 of the invention: MCF-7 (FIG. 7A), EL4 (FIG. 7B), HT-29 (FIG.7C), SW620 (FIG. 7D) and HepG2 (FIG. 7E). Given the above, itdemonstrates that the compounds and ACM Extract of the invention haveinhibition effect on various types of tumor cells.

Analysis of all New Compounds (1, 2 and 3) from ACM EtOH Extract by HighPerformance of Liquid Chromatography Method

Purpose: In order to measure the amount of all new compounds (1, 2 and3) from ACM EtOH Extract, High Performance of Liquid Chromatography wasemployed as our routine quality control procedures.

Preparation for ACM EtOH Extract Sample:

1) By using digital balance precisely weight 20,000 (g) of samplepowders in a graduated media lab bottle with 100 mL of 95% alcohol, anddo not screw the lid tightly on.2) Place above step of the sample bottle in ultrasonic water bath 10minutes.3) Pour liquid samples to centrifuge tubes, and then place those samplesin a centrifuge remove crude particle, under condition of 6500 rpm/5minutes.4) Filter liquid layer with filter paper, No. 1.5) Concentrate filtering solution by rotary vacuum evaporator untilappear a thick, yellowish liquid, alcohol free.6) Repeat three times of step 1 to 5, and then collect all extractproduct (total ACM EtOH Extract=4.60 g). Calculate yield.

Application By Water HPLC, Model 2690: 1) Column: Reverse Phase C18

2) Mobile Phase: MeOH, H₂O, acetonitrile

3) Injection vol: 20 μL

4) Detection: Photodiode Array Detector 996 on wavelength 254 nm5) Preparation 1,000 (g) ACM EtOH Extract sample in 10 mL of alcohol forHPLC analysis*:

Results: according to HPLC analysis, the extract product contains purecompound 1, 2, and 3 was showed in following TABLE 4

TABLE 4 Standard Name Compound 1 Compound 2 Compound 3 For ingredientsof three standard compounds: weight 0.0100 (g) in 1 mL of alcoholConcentration 0.01 0.01 0.01 (g/mL) Peak Area 49,315,783 129,327,136136,255,406 Retention time 134.8 124.3 119.8 (min) *For ACM EtOH Extractsample: weight 1.000 (g) in 10 mL of alcohol Concentration 8.59 × 10⁻³5.59 × 10⁻⁴ 1.659 × 10⁻² (g/mL) Peak Area 42,374,766 7,226,937226,102,223 Percentage yield % 8.59 0.559 16.59 (w/w)

Therefore, the total weight of compounds 1, 2 and 3 is 5.92% by weightin ACM sample.

Tests for ACM-EtOH Extract Materials and Equipment Test Substances andDosing Pattern

Test substance was administered orally at an initial dose of 1000 mg/kgfor all in vivo assays in a vehicle of 2% Tween 80. Time of observationfor each assay was described in methods.

Animals

Male or female ICR mice, Wistar-Okamoto derived male spontaneouslyhypotensive rat (SHR), Wistar and Long Evans derived rats provided byMDS Pharma Services Taiwan Ltd. were used. Space allocation for animalswas as follows: 29×18×13 cm for 10 mice, 45×23×21 cm for 6 rats, and45×23×21 cm for 3 guinea pigs. Mice and rats were housed in APEC^(R)cages. The immunocompetent C57BL/6J male mice, 6-8 weeks age, weighing21±2 gm were also used in this study and provided by National TaiwanUniversity Animal Center. The animals were housed in IndividuallyVentilated Cages Racks (IVC racks, 36 Mini Isolator System). Each cagewas sterilized by autoclave and contained 5 mice (in 26.7×20.7×14 cm).All animal were maintained in a controlled temperature (21-23° C.) andhumidity (60%-70%) environment with 12 hour light dark cycles for atleast one week in the laboratory prior to use. Free access to standardlab chow (LabDiet Rodent Diet and Guinea Pig Diet, PMI NutritionInternational, USA) and tap water was granted.

Cell Line and Culture Media

The murine melanoma cell line, B16-F0 (ATCC CRL-6322), was purchasedfrom American Type Culture Collection and Dulbecco's Modified Eagle'sMedium (GIBCO, USA) was used as culture medium. The tumor cells wereincubated in an atmosphere containing 5% CO₂ at 37° C.

Chemicals

General: Distilled Water (In-house), Dimethyl Sulfoxide (DMSO, Merck,Germany), Isotonic Sodium Chloride Solution (Sintong Chemical IndustryCo. Ltd., R.O.C.), magnesium Sulfate (MgSO₄.7H₂O, Wako, Japan),Meclofenamate Sodium (Sigma, USA), Methylcellulose (Signa, USA), SodiumHydroxide (NaOH, Wako, Japan), Phosphate Buffered Saline (Sigma, USA)and Tween 80 (Wako, Japan).

Reagents

Glicose-HA assay kit (Wako, Japan), Alanine aminotransferase (ALT) assaykit (Wako, Japan), Aspartate aminotransferase (AST) assay kit (Wako,Japan), T-Cholesterol-HA and HDL assay kit (Wako, Japan), Hemolynac 3Hemolys (Nihon Koden, Japan), Isotonic 3 Diluent (Nihon Koden, Japan).

Equipment

General Use: Animal Case (ShinTeh, R.O.C.), Beaker 250 ml and 1000 ml(Kinmax, USA), disposable syringe (1 ml, Top Corporation, Japan),Forceps stainless (klappencker, Germany), Mouse scale #Z-40 (Taconic,USA), needle for oral administration (Natsune, Japan), Needle Hypodermic23 G×1″ (Top Corporation, Japan), pH Meter (Suntex, USA), Rat scale 500g±2 g (Chien-chun, ROC), syringe Glass 1 ml, 2 ml and 5 ml (Mitsuba,Japan), and Scissors Stainless (Klappencker, Germany).

Methods and Results:

Cholinergic Agonism, Central/Peripheral, Lippmann, W. and Pugsley, T.A., Arch. Int. Pharmacodyn. 227:324 (1977). Test substance wasadministered orally to a group of 3 Wistar derived male or female ratsweighing 150±20 g. During the subsequent 30-60 minute period, the numberof animals exhibiting more than 10 seconds of chewing behavior (mouthand/or tongue movements) measured cumulatively and the number of animalsexhibiting salivation or exhibiting salivation were noted. Positiveresponses observed in 2 or more (2) of 3 rats indicates possible centralcholinergic activity and peripheral cholinergic activity.

TABLE 5 Result of Cholinergic Agonism, Central/peripheral in RatsCentral Peripheral Treatment Route Dose N Chewing Score Salivation ScoreVehicle PO 10 ml/kg 1 − − 2 − − 3 − 0/3 − 0/3 ACM-EtOH Extract PO 1000mg/kg 1 + − 2 + − 3 + (3/3) − 0/3 300 mg/kg 1 − − 2 − − 3 + 1/3 − 0/3Arecoline-HBR IP 30 mg/kg 1 + + 2 + + 3 + (3/3) + (3/3)

Vehicle and test substances were administered orally (PO) while thepositive reference compound was injected intraperitoneally (IP). Duringthe subsequent 30-60 minute period, the number of animal exhibiting morethan 10 seconds of chewing behavior (mouth and/or tongue movements)measured cumulatively or exhibiting salivation were noted. Positiveresponses observed in 2 or more (2) of 3 rats indicates possiblecholinergic activity or peripheral cholinergic activity.

Cardiovascular, Blood Pressure and Heart rate (SHR 0, 1, 2, 4 hrs) (Yen,T. T., et al., Life Sci. 22: 359, 1978). Groups of 3 Wistar-Okamotoderived male spontaneously hypertensive rats (SHR) weighing 250±20 gwere used; the mean systolic blood pressure was 200±20 and heart rate400±30 beats/min. Blood pressure and heart rate were recorded indirectlyby tail cuff method in a temperature controlled environment (32±1° C.)before (0 time) and 1, 2 and 4 hours after oral administration of testsubstance or vehicle. A reduction in systolic pressure by 10 percent ormore (>10%), or decrease in heart rate by 20 percent or more (≧20%), ateach measured time interval relative to 0 time, is consideredsignificant.

TABLE 6 Result of Cardiovascular, Blood Pressure (SHR 0, 1, 2, 4 Hours)in Rats % Control (from 0 times) Treatment Route Dose N 1 Hour 2 Hours 4Hours Vehicle PO 10 ml/kg 1 100  96 90 2 97 100  91 3 90 92 92 Ave. 9696 91 ACM-EtOH PO 1000 mg/kg 1 78 85 71 Extract 2 86 89 80 3 89 89 89Ave. (84) (88) (80) Clonidine PO 0.1 mg/kg 1 71 67 71 2 95 86 88 3 72 8569 Ave. (79) (79) (76)

TABLE 7 Result of Cardiovascular, Heart Rate (SHR 0, 1, 2, 4 Hours) %Control (from 0 times) Treatment Route Dose N 1 Hour 2 Hours 4 HoursVehicle PO 10 ml/kg 1 87 100 99 2 116  103 107 3 108  104 121 Ave. 104 102 109 ACM-EtOH PO 1000 mg/kg 1 98 93 95 Extract 2 81 100 88 3 83 78 92Ave. 87 90 92 Clonidine PO 0.1 mg/kg 1 62 97 112 2 84 87 104 3 68 86 78Ave. (71) 90 98

SHR with systolic blood pressure of 200±20 mmHg and heart rates of400±50 Beats/min were used. Blood pressure was recorded indirectly vialtail cuff at 0 time (before) and 1, 2 and 4 hours after oraladministration of vehicle or test substance. A reduction in bloodpressure by 10 percent or more (10%), or decrease in heart rate by 20percent or more (≧20%) at each measurement time point relative to 0time, shown in parenthesis, is considered significant.

Vehicle  10 ml/kg 0 time 229 and 403 beats/minute as 100%. ACM-EtOH 1000mg/kg 0 time 223 and 452 beats/minute as 100%. Extract Clonidine   0.1mg/kg 0 time 228 and 379 beats/minute as 100%.

TABLE 8 Result of Cardiovascular, Blood Pressure (SHR 0, 1, 2, 4 Hours)in Rats % Control (from 0 times) Treatment Route Dose N 1 Hour 2 Hours 4Hours Vehicle PO 10 ml/kg 1 94 97 97 2 88 97 94 3 94 97 103  Ave. 92 9798 ACM-EtOH PO 300 mg/kg 1 111  102  103  Extract 2 94 84 100  3 112 110  112  Ave. 106  99 105  Clonidine PO 0.1 mg/kg 1 86 73 81 2 63 73 903 62 68 80 Ave. (70) (71) (85)

TABLE 9 Result of Cardiovascular, Heart Rate (SHR 0, 1, 2, 4 Hours) %Control (from 0 times) Treatment Route Dose N 1 Hour 2 Hours 4 HoursVehicle PO 10 ml/kg 1 82 85 84 2 88 115 102 3 109  111 119 Ave. 93 104102 ACM-EtOH PO 300 mg/kg 1 97 96 92 Extract 2 105  108 98 3 85 96 82Ave. 96 100 91 Clonidine PO 0.1 mg/kg 1 77 85 102 2 78 78 100 3 62 94104 Ave. (72) 86 102

SHR with systolic blood pressure of 200±20 mmHg and heart rates of400±50 beats/min were used. Blood pressure was recorded indirectly vialtail cuff at 0 time (before) and 1, 2 and 4 hours after oraladministration of vehicle or test substance. A reduction in bloodpressure by 10 percent or more (10%), or decrease in heart rate by 20percent or more (≧20%) at each measurement time point relative to 0time, shown in parenthesis, is considered significant.

Vehicle  10 ml/kg 0 time 220 and 410 beats/minute as 100% ACM-EtOH 300mg/kg 0 time 205 and 446 beats/minute as 100% Extract Clonidine  0.1mg/kg 0 time 235 and 417 beats/minute as 100%

-   1. Schurr et al., Cholesterol, Serum (Total HDL, total/HDL, Ratio),    Diet-Induced, Atherosclerosis Drug Discovery. Plenum, N.Y., pp.    215-229, 1976)

Groups of 5 ICR derived male mice weighing 22±2 g were kept on a highfat diet (g/100 g: coconut oil, 8; cholesterol, 1.0; cholic acid, 0.3;lard 2; standard chow 88.7) for 7 days to induce hypercholesterolemia.Test substance was administered orally on days 5, 6 and 7. After fastingovernight, serum was obtained from each mouse and assayed for totalcholesterol (Total), high density lipoprotein (HDL) and percent changein Total/HDL. A decrease of 20 percent or more (20%) in serum Total orincrease of 20 percent or more (≧20%) in serum HDL or decrease of 40% ormore (≧40%) in the Total/HDL ratio relative to vehicle treated controlanimals is considered significant.

TABLE 10 Result of Cholesterol, (Total/HDL, Total/HDL Ratio),Diet-Induced in Mice Total HDL Total/HDL Treatment Route Dose N Indiv. %Dce Indiv. % Dce Indiv % Dce Vehicle PO 10 ml/kg × 3 1 361 70 5.16 2 31682 3.85 3 379 79 4.80 4 392 78 5.03 5 367 86 4.27 Ave. 363 — 79 — 4.59 —ACM-EtOH PO 1000 mg/kg × 3 1 420 117 3.95 Extract 2 327 115 2.84 3 332104 3.19 4 363 98 3.70 5 294 117 2.51 Ave. 347  4 110 (39) 3.15 31 PO300 mg/kg × 3 1 370 66 5.61 2 301 65 4.63 3 217 74 2.93 4 379 76 4.99 5328 98 3.35 Ave. 319 12 76 −4 4.20  8 Benzafibrate PO 100 mg/kg × 3 1230 91 2.53 2 214 120 1.78 3 225 133 1.69 4 231 123 1.88 5 242 97 2.49Ave. 228 (37) 113 (43) 2.02 (56)

Vehicle, test substance or reference positive compound was administeredorally (PO) on days 5, 6 and 7 after being fed a high cholesterol diet.Twenty-four hours after the third dose, the overnight-fasted testanimals were sacrificed for assessing serum total cholesterol (Total)and high density lipoprotein (HDL). Decrease of 20 percent or more (20%)in serum Total or increase of 20 percent or more (20%) in serum HDL ordecrease of 40% or more (40%) in the Total/HDL ratio is consideredsignificant.

-   2. Hepatic Injury, D-Galactosamine (Wrobel et al., J. Med. Chem. 41:    1084, 1998)

Groups of 5 Wistar derived male rats weighing 200±20 g were used. Eachanimal was treated with a single injection of D-galactosamine (500mg/kg, IP) Test substance was administered orally at 0.5 hour before and4 hours as well as 8 hours after D-galactosamine administration andanimals were sacrificed 24 hours later. Serum alanine aminotransferase(ALT) and aspartate aminotransferase (AST) levels were measured by anoptimized UV method with HITACHI automatic analyzer (model 7050).Reduction in ALT or AST activity by 30 percent or more (30%), relativeto the vehicle treated control animal indicates significant protection.

TABLE 11 Result of Hepatic Injury, Galactosamine in Rats Serum ALT (X ±Serum AST (X ± SEM) SEM) Dec. Dec. Treatment Route Dose N U/L % U/L %Vehicle PO 10 mg/kg × 3 1 816 1628 2 1044 1716 3 652 888 4 656 828 5 644956 X 762.4 — 1203.2 — SEM 77.4 193.0 ACM-EtOH PO 1000 mg/kg × 3 1 364516 Extract 2 376 532 3 596 672 4 452 524 5 336 356 X 424.8 (44) 520.0(57) SEM 46.9 50.1 300 mg/kg × 3 1 460 852 2 656 880 3 640 876 4 7521004 5 536 692 X 608.8 20 860.8 28 SEM 50.6 49.8 Guanine PO 300 mg/kg ×3 1 508 656 2 532 912 3 412 776 4 436 652 5 636 1028 (34) (33)

Test substance and vehicle were administered orally at 0.5 hour beforeand 4, 8 hours after a single dose of galactosamine (500 mg/kg, IP). Therats were sacrificed 24 hours after galactosamine injection and the ALTand AST values were determined A reduction of 30% in the ALT and ASTrelative to the vehicle group is considered significant.

-   3. Winter et al., Inflammation, Carrageenan, Proc. Soc. Exp. Biol.    Med. 111:544, 1962.

A group of 3 Long Evans derived male or female overnight fasted ratsweighing 150±20 g was fasted overnight prior to study. Test substancewas administered orally one hour before right hind paw receivedinjection of carrageenan (0.1 ml of 1% suspension intraplantar). Hindpaw edema, as a measure of inflammation, was recorded 3 hours aftercarrageenan administration using a plethysmometer with water cell (25 mmdiameter). Reduction of hind paw edema by 30 percent or more (30%)indicates significant acute anti-inflammatory activity.

TABLE 12 Result of Inflammation, Carrageenan in Rats Paw Volume (×0.01ml) Treatment Route Dose N R.P. L.P. Diff % Inhibition Vehicle PO 10ml/kg 1 194 103 91 2 202 108 94 3 199 104 95 Ave. 198 105 93 — ACM-EtOHPO 1000 mg/kg 1 146 101 45 Extract 2 147 95 52 3 160 104 56 Ave. 151 10051 (45) Aspirin PO 150 mg/kg 1 152 102 50 2 146 102 44 3 163 106 57 Ave.154 103 50 (46)

TABLE 13 Result of Inflammation, Carrageenan in Rats Paw Volume (×0.01ml) Treatment Route Dose N R.P. L.P. Diff % Inhibition Vehicle PO 10ml/kg 1 193 105 88 2 198 107 91 3 199 102 97 Ave. 197 105 92 — ACM-EtOHPO 300 mg/kg 1 195 104 91 Extract 2 187 103 84 3 196 103 93 Ave. 193 10389  3 Aspirin PO 150 mg/kg 1 146 103 43 2 149 101 48 3 169 104 65 Ave.155 103 52 (43)

Vehicle or test substance was administered to overnight fasted rats onehour before right hindpaw (R.P.) injection of carrageenan (0.1 ml of 1%suspension intraplantar); the left hindpaw (L.P.) was not injected.Reduction of hindpaw edema by 30 percent or more (30%), shown inparenthesis, indicates significant acute anti-inflammatory activity.

-   4. Tumor, Syngeneic, Melanoma, B16-F0 Cell (Farrugia C A and Groves    M J. Anticancer Research 19: 1027-1032, 1999)

Groups of 5 immunocompetent (6-8 weeks old), pathogen-free (SPF)C57BL/6J male mice bred in an animal isolator (IVC racks) under specificpathogen free (SPF) condition were used. Viable B16-F0 murine melanomacells (ATCC CRL-6322, 1.0×10⁵ in 0.2 ml), syngeneic for C57BL/6J mice,were injected subcutaneously into dorsal side of experimental mice.Treatment begins 24 hours after tumor inoculation and test compound wasadministered daily by oral gavage for 21 days, or less when overt signsof toxicity are seen. The mice were monitored for body weight, tumorsize and survival starting from day 1 to day 22. Moreover, the testedmice were monitored for survival till the end of the study on day 45.

Tumor weight (mg) was estimated according to the formula for a prolateellipsoid: length (mm)×[width (mm)]²×0.5, assuming specific gravity tobe one and r to be three. Tumor growth in compound treated animals wascalculated as T/C (Treatment/Control)×100%; a value of T/C 42% wasconsidered significant in demonstrating antitumor activity. The meansurvival time of T/C (Treatment/Control) is 125% is also consideredsignificant in demonstrating antitumor activity.

TABLE 14 Result of Tumor, Syngeneic, Melanoma B16-F0 Cell Tumor Weight(mg) and % T/C, Mean ± SEM Treatment Route Dose N Day 1. T/C(%) Day 8T/C(%) Day 11 T/C(%) Vehicle PO 10 ml/kg × 21 1 0 0 60 2 0 39 298 3 0 049 4 0 54 541 5 0 21 117 0 100 23 ± 11 100   213 ± 93  100  ACM-EtOH PO1000 mg/kg × 21 1 0 0 0 Extract 2 0 0 0 3 0 0 0 4 0 0 14 5 0 0 32 0 1000 ± 0 0* 9 ± 6  4* Mitomycin IP 2 mg/kg × 6 1 0 0 0 2 0 0 64 3 0 0 0 4 00 68 5 0 0 41 0 100 0 ± 0 0* 34 ± 15 16*

TABLE 15 Result of Tumor, Syngeneic, Melanoma B16-F0 Cell Tumor Weight(mg) and % T/C, Mean ± SEM Treatment Route Dose N Day 15 T/C(%) Day 18T/C(%) Day 22 T/C(%) Vehicle PO 10 ml/kg × 21 1 211 746 2054 2 657 15972870 3 216 669 1419 4 835 2455 3688 5 240 726 1682  432 ± 131 100  1239± 349  100  2343 ± 416 100  ACM-EtOH PO 1000 mg/kg × 21 1 49 280 913Extract 2 62 630 1545 3 388 1079 2560 4 148 435 1514 5 229 535 1637 175± 62 41* 592 ± 135 48  1634 ± 265 70  Mitomycin IP 2 mg/kg × 6 1 36 256437 2 136 849 1248 3 0 0 0 4 213 525 663 5 207 327 Died 119 ± 44 27* 391± 142 32*  587 ± 260 25*

Vehicle and test substance were administered to test animals every dayat 24 hours after tumor cells implantation for a total of 21 doses.Concurrently, the reference compound, mitomycin, was administered IPtwice a week for a total of 6 doses. Tumor size was measured andrecorded twice a week for a period of 22 days. Tumor growth inhibitionwas calculated as T/C (treatment/control)×100. A T/C value of 42% wasconsidered significant in demonstrating antitumor activity.

TABLE 16 Result of Tumor, Syngeneic, Melanoma B16-F0 Cell Body Weight(g), Mean ± SEM Treatment Route Dose N Day 1 Day 8 Day 11 Day 15 Day 18Day 22 Vehicle PO 10 ml/kg × 21 1 21 20 20 21 22 25 2 22 22 21 22 26 303 21 21 20 20 22 21 4 21 20 20 20 21 24 5 22 21 20 19 20 23 21.4 ± 0.220.8 ± 0.4 20.2 ± 0.2 20.4 ± 0.5 22.2 ± 1.0 24.6 ± 1.5 ACM-EtOH PO 1000mg/kg × 21 1 20 21 21 22 22 23 Extract 2 20 19 19 21 22 24 3 20 18 18 1921 26 4 21 20 19 21 20 21 5 20 20 21 22 23 25 20.2 ± 0.2 19.6 ± 0.5 19.6± 0.6 21.0 ± 0.5 21.6 ± 0.5 23.8 ± 0.9 Mitomycin IP 2 mg/kg × 6 1 25 2526 25 24 27 2 22 22 22 25 27 30 3 19 21 21 21 22 21 4 22 22 22 24 26 275 19 20 19 20 21 Died 21.4 ± 1.1 22.2 ± 0.8 22.0 ± 1.1 23.0 ± 1.0 24.0 ±1.1 26.3 ± 1.9

Vehicle and test substance were administered to test animals every dayat 24 hours after tumor cells implantation for a total of 21 doses.Concurrently, the reference compound, mitomycin, was administered IPtwice a week for a total of 6 doses. Tumor size was measured andrecorded twice a week for a period of 22 days. The Student's t test wasused to determine the significant difference in the change of bodyweight between test compound and vehicle control group.

TABLE 17 Result of Tumor, Syngeneic, Melanoma B16-F0 Cell Survival Time(day), Mean ± SEM Days of Post- Treatment Route Dose N treatment T/C (%)Vehicle PO 10 ml/kg × 1 23 21 2 28 3 30 4 28 5 27 27.2 ± 1.2 100 ACM-EtOH PO 1000 mg/kg × 1 44 Extract 21 2 31 3 25 4 42 5 32 34.8 ± 3.6128* Mitomycin IP 2 mg/kg × 6 1  45^(a) 2 28 3  45^(a) 4 30 5 22 34.0 ±4.7 125* ^(a)The animal did not die through day 45 and the survival daywas served as 45 days. The treated mice were monitored for survivalthrough the end of the study on day 45 or the day when test animal died.The mean survival time of T/C (Treatment/Control) 125% is alsoconsidered significant in demonstrating anti-tumor activity.

Discussion:

ACM-EtOH Extract, administered orally (PO), in accordance with in-houseestablished criteria, caused significant activities in the followingmouse and rat assays:

Central cholinergic agonism at 1000 mg/kg in rats; a minimum andnon-significant agonism was seen a 300 mg/kg; no significant agonism orantagonism on peripheral cholinergic nerve was seen at 1000 mg/kg (TABLE5)

Decrease in systolic blood pressure (16%, 12% and 20% at respective 1, 2and 4 hours observation time points vs. 100% with 0 time) and associatedmoderate but non-significant decrease in heart rate at 1000 mg/kg inspontaneously hypertensive (SH) rats (TABLES 6 and 7); dose of 300 mg/kgdid not cause significant changes in systolic blood pressure nor theheart rate (TABLES 8 and 9)

Increase in high density lipoprotein (HDL, 39% over vehicle control) at1000 mg/kg in diet-induced mice (TABLE 10); the associate totalcholesterol (Total) did not change significantly, while the HDL/totalratio was decreased to near significant 31%; dose of 300 mg/kg did notcause significant changes in Total, HDL and HDL/Total ratio (TABLE 10).

Hepatoprotection (44% decrease in ALT and 57% decrease in AST vs.vehicle control) from galactosamine induced hepatic injury in rats at1000 mg/kg×3; moderate decrease of 20% in ALT and of 28% in AST at 300mg/kg×3 was seen (TABLE 11).

Anti-inflammation (45% inhibition vs. vehicle control) versuscarrageenan-induced paw edema in rats at 1000 mg/kg (TABLE 12); thelower level of 300 mg/kg did not demonstrate significant activity (3%inhibition vs. vehicle, TABLE 13).

Anti-tumor activity in syngeneic melanoma B16-F0 cell for C57BL/6J miceon day 8, 11 and 15 (TABLES 14 and 15) as well as prolongation in animalsurvival time at 1000 mg/kg (TABLE 17); animal body weight did notchange significantly (TABLE 16).

Tests for ACM, ACM-ETOH Extract and Compound 3 of the Invention

Nine groups of ICR derived male mice (weighing 22±2 g) of 5 each wereused. Each animals was challenged with a single dose of carbontetrachloride (CCl₄, 0.1 ml/kg in 50% olive oil, PO). The test substanceof ACM at doses 300 and 1000 mg/kg or compound 3 of the invention atdoses 30, 100 and 300 mg/kg were administered orally at 30 minutesbefore and 4, 8 hours after CCl₄ challenge; whereas ACM-ETOH Extract at300 and 1000 mg/kg administered orally was pretreated one day (twice aday) and 30 minutes before and 4, 8 hours after carbon tetrachloride.The animals were sacrificed 24 hours after CCl₄. Alanineaminotransferase (ALT) and Aspartate aminotransferase (AST) levels weremeasured by optimized UV method using a HITACHI automatic analyzer(model 7050). A reduction of ALT or AST levels by 30 percent or greater(30%), relative to the vehicle group, indicating significant protectionfrom hepatic injury.

Results

TABLE 18 Assay Hepatic Injury, Carbon Tetrachloride, in Mice ALT ASTTreatment Route Dose N U/L Dec. % U/L Dec. % Vehicle (2% PO 10 ml/kg × 31 3936 2056 Tween 80) 2 3456 1856 3 3712 1528 4 2560 1328 5 2968 1696 X3326  0 1693  0 SEM 250 126 ACM PO 1000 mg/kg × 3 1 1440 888 2 2720 15203 2272 1328 4 1272 792 5 1320 880 X 180.5 (46) 1082 (36) SEM 292 144 300mg/kg × 3 1 2336 1256 2 1552 1072 3 3720 1512 4 3816 2336 5 3952 2792 X3075  8 1794 −6 SEM 480 330 ACM-EtOH PO 1000 mg/kg × 5 1 1936 1232Extract 2 1528 768 3 1896 1136 4 2752 1656 5 2472 1592 X 2117 (36) 127725 SEM 219 162 300 mg/kg × 5 1 1656 976 2 3536 1712 3 2328 1808 4 17361416 5 1792 888 X 2210 (34) 1360 20 SEM 352 187 Compound 3 PO 300 mg/kg× 3 1 1368 776 2 1576 896 3 1440 896 4 2728 1352 5 2720 1728 X 1966 (41)1130 (33) SEM 311 179 100 mg/kg × 3 1 3200 2256 2 4576 2976 3 2512 15364 2728 1552 5 3696 1600 X 3342  0 1984 −17  SEM 370 282 30 mg/kg × 3 14296 2136 2 3696 2288 3 2152 1096 4 2400 1792 5 4256 2496 X 3360 −1 1962−16  SEM 457 245 Silymarin PO 100 mg/kg × 3 1 2856 1296 2 1832 1152 31296 952 4 2792 1072 5 2728 1336 X 2301 (31) 1162 (31) SEM 3136 71

Discussion

ACM, ACM-EtOH Extract and compound 3 of the invention were evaluated forpossible protective activity from hepatic injury induced by carbontetrachloride in ICR mice. The test substance of ACM at doses 300 and1000 mg/kg and compound 3 of the invention at doses 30, 100 and 300mg/kg were administered orally to test animals 0.5 hour before and 4, 8hours after CCl₄ challenge. For ACM-EtOH Extract at 300 and 1000 mg/kg,2 times (b.i.d.) of treatment (9:00 AM and 16:00 PM) were done 1 daybefore CCl₄ and followed 0.5 hr before and 4, 8 hours after CCl₄challenge (5 dosing in total). The degree of hepatic injury wasdetermined by increase in serum alanine aminotransferase (ALT) andaspartate aminotransferase (AST) levels relative to the vehicle treatedanimals. ACM at 1000 mg/kg×3 and compound 3 of the invention at 300mg/kg×3 caused a significant reduction of ALT (46% and 41%) and AST (36%and 33%) relative to the vehicle treated animals. Simultaneously,ACM-EtOH Extract at 300 and 1000 mg/kg×5 also caused significantreduction in ALT (36% and 34%) and AST (25% and 20%).

Concurrently tested silymarin (100 mg/kg×3, IP) showed significantlyreduction of ALT (31%) and AST (31%) relative to the vehicle treatedgroup. It is concluded that ACM, ACM-EtOH Extract and compound 3 of theinvention possess the ability of significant hepatoprotectant activityin a mouse CCl₄ model.

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The cell lines, embryos,animals, and processes and methods for producing them are representativeof preferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Modifications therein andother uses will occur to those skilled in the art. These modificationsare encompassed within the spirit of the invention and are defined bythe scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitations,which are not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

1. A compound having the formula

wherein R₁ is C₁₋₁₀ alkyloxy, C₂₋₁₀ alkenyloxy, or C₂₋₁₀ alkynyloxy; andR₂ is H, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl or C₂₋₁₀ alkynyl, provided that ifR₁ is C₁₋₁₀ alkyloxy, R₂ is not H, and if R₁ is C₁ alkyloxy, R₂ is notC₁ alkyl.
 2. The compound of claim 1, wherein R₁ is C₂₋₆ alkenyloxy orC₂₋₆ alkynyloxy.
 3. The compound of claim 2, wherein C₂₋₆ alkenyloxy issubstituted with C₁₋₆ alkyl.
 4. The compound of claim 1, wherein R₂ isisobutyl.
 5. The compound of claim 1, which is3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-dione.